Project Summary ? Project 3 Project 3 will investigate the physical and metabolic constraints of cancer cell invasion. Cancer cell invasion from the primary tumor into surrounding tissues is a crucial step of the metastatic cascade, which is responsible for the vast majority of all cancer deaths. Invading cancer cells can use a number of different `migration modes' for this process. As cells can dynamically switch between these modes, therapeutic attempts to target specific migration mechanisms have had limited clinical success. Gaining an improved understanding of the physical and biological mechanisms that govern the toggling between different migration modes could provide new clues for more robust therapeutic interference to reduce or eliminate metastasis. One important factor that has not been examined previously is how the metabolic status of individual cells can determine their migration mode and metastatic spreading. As cancer cells pass through tight interstitial spaces and metabolite- poor regions in vivo, they face substantial physical and metabolic challenges. We propose that invading cancer cells must expend significant energy to penetrate such environments and adopt migration modes that minimize metabolic cost. Cancer cell metabolism can also impact cellular structure and composition by fueling biosynthetic pathways, which could alter cell surface architecture and nuclear deformability, and thereby promote migration through tight spaces. The proposed research will utilize the project team's complementary expertise in cell migration, subcellular biomechanics, intravital microscopy, and metabolic pathways and interference. Aim 1 addresses how the physical microenvironment and adhesion/friction between the cell and the extracellular environment determine energy consumption during different types of cell migration. Aim 2 will investigate how physical factors intrinsic to the cell, particularly nuclear deformability and the physical properties of the cell surface, modulate metabolic cost and migration efficiency in physiologically relevant environments. It will further determine how metabolic reprogramming in cancer cells affects these cellular mechanical properties and thereby modulates migration efficiency. Aim 3 will investigate the plasticity of cancer cell migration, both in response to varying physical and metabolic challenges and to pharmacological interference. The experimental work in each aim will be complemented by modeling of cancer cell metabolism and physical interaction with the microenvironment, with the objective to predict outcomes of therapeutic metabolic interventions and to identify strategies to counter adaptive responses. Project 3 will interact with both Cores. The Tissue Microfabrication Core will provide biological samples and platforms for migration assays; the Biophysics & Metabolic Imaging Core assists with metabolic analysis and imaging platforms. Projects 1 and 2 will contribute data and insights for a comprehensive computational modeling framework of cell metabolism in migration; Project 2 will additionally provide tumor microvesicles for functional evaluation.